Although western blotting is a common technique, there are still many issues that arise from the protein transfer step. These difficulties include the introduction of air bubbles when placing gels on membranes, and the gels often tear when they are transferred from a precast or other casted gel setting to a separate protein transfer membrane, especially in the case of thinner gels that are employed to reduce the amount of protein needed. These complications can be devastating when the availability of the protein sample is limited, no more protein is available, or when the protein is a clinical specimen. The source of many of the problems with the prior art can be traced to the need to remove the gel from the gel cassette in order to accomplish the transfer step. It has not been possible to eliminate this step in the prior art because the gel cartridges are made of materials that are non-conductive/insulative and are used to support the gels. Additionally, researchers often prefer to watch the electrophoretic step progress, which has limited the motivation to invent electrophoretic gel systems incorporating non-transparent conductive materials.
It would be desirable to have a one-step separation and transfer western blot apparatus and method of separating and transferring proteins that utilize a single combination of a precast gel and protein transfer membrane. Hence, it would be advantageous to have a means to avoid the introduction of air bubbles, and to avoid the tearing of gels during transfer.
There have been many attempts to simplify the separation and transfer of proteins for analysis using various types of apparati and techniques. U.S. Pat. No. 4,994,166 to Fernwood et al. describes a single apparatus for slab gel electrophoresis and blotting, both of which are performed in a single tank cell, which contains separation electrodes along opposing vertical walls, and blotting electrodes arranged horizontally above and below the level of gel placement. The cell is operated in separatory and blotting modes, in which separatory and blotting electrodes are separately energized. Fernwood requires porous gel supports to allow the electric field to pass through the membrane. In addition, the top plate transfer electrode must be removed from contact with the buffer solution during the protein separatory phase.
U.S. Pat. No. 5,102,524 to Dutertre describes a multiple electrophoresis method, where different sets of electrodes are used in a two-step process to first separate biomolecules and then to transfer them to a deposition membrane.
U.S. Pat. No. 5,593,561 to Cognard describes a multiple electrophoresis method for controlled migration of biomolecules and transfer thereof to a membrane in a vessel, containing a plurality of parallel elongated electrodes. The first electric field, established between electrodes, provides means for macromolecular separation in a gel, and the second electric field, perpendicular to the first, provides means for transferring the biomolecules onto the membranes. In the described method, electrodes and transfer membranes are first assembled in the vessel, which is then filled with gel. After the separation of biomolecules in the gel, and the proteins are transferred to the membrane, the gel is liquefied, dissolved, or decomposed, which allows for the removal of the membrane. Cognard's invention is for use without prefabricated gel and membrane combination units.
U.S. Pat. No. 8,173,002 to Margalit describes a dry blotting system to transfer proteins onto a transfer membrane. The system does not include an electrophoresis device, so the device does not allow the user to visualize the separation and transfer phases in a single device. The device requires the user to transfer the gel to a transfer membrane on the blotting device. Margalit teaches the use of electrically conducting polymers, but not in combination with a single device that both separates proteins and transfers the proteins to a transfer membrane.
U.S. Patent Appl. Pub. No. 2006/0042951 to Ohse discloses an apparatus to separate and transfer proteins via the use of a fine groove, a transferring electrode, and a transparent conductive material having a thickness of approximately 0.1 μm. The apparatus includes a pair of separating electrodes for causing a substance in a sample to move along a passage, and a pair of transferring electrodes for causing the substance in the sample to be transferred to the capturing material by electrophoresis. The conductive material is not capable of serving as the support structure due to its thickness of approximately 0.1 μm, which would not have sufficient strength to serve effectively as the supporting walls for a gel. The separation and blotting is performed in an electrophoresis buffer and does not make use of a gel slab or gel slab assembly, which are commonly used for western blots.
U.S. Pat. No. 6,602,391 to Serikov discloses an apparatus and method for capillary separation of biomolecules and post-separation blotting. However, Serikov does not disclose the use of a slab gel where the user can view the separation of biomolecules and transfer the macromolecule to a blotting membrane for western blotting.
Conductive polymers have previously been described, but not in conjunction with electrophoresis and blotting. Ates et al. Describes various applications of conducting polymers in “Conducting Polymers and their Applications” (Current Physical Chemistry, 2012, 2, 224-240). International Pat. Appl. No. PCT/EP2013/065163 to Jung discloses a conductive polymer composition and transparent electrode for an antistatic layer. International Pat. Appl. No. PCT/KR2008/002236 to Kim discloses a conductive polymer for use as a transparent electrode and a method of fabricating the electrode using an ink jet spray method. U.S. patent application Ser. No. 13/616,804 to Kim et al. discloses a transparent panel and method of manufacturing a transparent panel where a conductive polymer layer is formed to make a transparent electrode. U.S. patent application Ser. No. 15/017,540 to Woodham discloses an apparatus and method for using a gel transfer combination having transparent conductive polymers and to separate proteins during an electrophoresis phase and thereafter transfer proteins to a transfer membrane after electrophoresis without transferring the gel from an electrophoresis apparatus to a separate protein blotting transfer apparatus. Opaque conductive polymers also have previously been described, but not in conjunction with electrophoresis and blotting. For example, U.S. Pat. No. 5,609,315 to Lepore describes electrically conductive opaque sheets made of polyimide and U.S. Pat. No. 4,702,371 to Hoshi describes electrically conductive portions of a plastic material to prevent electrostatic breakdown of electrical components such as those in integrated circuits.
One challenge in creating a single combination gel electrophoresis and protein transfer unit involves creating an apparatus where the user can visualize the degree of protein separation during electrophoresis and thereafter transfer the proteins to a blotting membrane without physically transferring the gel to the membrane. For such an apparatus to perform both electrophoresis and protein transfer, the plates must form the structural support for the gel, and also be able to transfer current through the gel supporting plates to a blotting membrane. The challenge is that the electrical current required to transfer proteins to the blotting membrane must run perpendicular to the current required to separate proteins during electrophoresis. Gel supporting plates that can be made rigid, transparent, and conductive would be ideal for use in such a gel and membrane combination unit.
One promising material that can provide rigidity, transparency, and conductivity are transparent metal compositions such as indium tin oxide (ITO). Compositions like ITO have been used in some applications where both conductivity and transparency are required, however, a considerable compromise must be made between conductivity and transparency. Likewise, transparent polymers such as conductive transparent plastics could be used, but there is also a compromise between conductivity and transparency. Furthermore, transparent conductive polymers are costly and may be cost prohibitive for commercial application such for the use in western blots.
Another problem with separating proteins within an electrophoresis gel is that if the user does not carefully monitor the protein separation phase, proteins may run off of the gel into the electrophoresis buffer. One way to prevent proteins from running off the gel is to have the electrophoresis power source on a timer so that after a pre-set amount of time has elapsed, the current shuts off. However, due to several variables (such as gel thickness and temperature), a timed shut-off may not allow the proteins to be optimally separated if the timer is set too short. If the timer is set too long, the proteins may run off the gel into the buffer. In addition, once the electrophoresis timer stops, proteins begin to diffuse within the gel. If the protein transfer step is not performed immediately after electrophoresis, protein bands may not be sufficiently defined.
Given the disadvantages of using a timer to end the electrophoresis phase, an alternative is to use an optical sensor that is capable of detecting the dye front of a sample loading buffer with known separation characteristics that is loaded into the wells of the gel as part of the protein running sample. When the optical sensor detects the dye front, the sensor communicates this information to the power source and shuts the current off, or controls the current in some other manner. Different types of electrophoresis power control devices have previously been described. One optical sensor linked power control device is described in PCT Appl. No. PCT/US2013/030220 to Asare-Okai et al. Asare-Okai discloses a controller with a sensor that is positioned adjacent to a gel matrix. The sensor includes a light source for emitting light into the gel matrix and a light detector disposed adjacent to the light sources for detecting light from the illuminated gel matrix. The controller is connected to a power source that provides a current across the electrophoresis phase electrodes. The controller is operable for turning off electrical power to the power source based on a change in the light emitted from the gel matrix due to migration of the tracking dye through the illuminated gel matrix.
U.S. Pat. No. 5,120,419 to Papp discloses a photoelectric electrophoresis controller. The controller is triggered by molecular dyes that are sensed by the photodetector when the dye reaches a predetermined position in the gel matrix, characterized by an observing photocell spaced from a reference photocell for comparison.
U.S. Pat. No. 5,268,568 to Lee discloses a marker dye band detector for gel electrophoresis using balanced light emitters. The device is capable of detecting a marker dye used in gel electrophoresis when the marker dye has reached a specific position in the gel. The device activates an alarm or shuts off the power source when the sensor detects the dye. Aare-Okai, Papp, and Lee do not disclose the use of optical sensors in a combination apparatus that can both separate and transfer proteins to a blotting membrane.
In view of the above limitations in the field, there currently exists a need in the industry for a device and associated method that can perform gel electrophoresis and protein transfer to a blotting membrane in one precast gel and transfer membrane combination unit.
All patents, patent applications, and non-patent references disclosed in the background and description of this application are hereby incorporated by reference for all purposes in their entireties.